Diagnostic regeant for hepatitis C virus infection

ABSTRACT

A diagnostic reagent for hepatitis C virus infection obtained by sensitizing a solid phase with HCV antigen and a conjugated antigen prepared by chemical bonding of HCV antigen and a carrier protein, and a method of diagnosing hepatitis C virus infection, which comprises adding the diagnostic reagent for hepatitis C virus infection to a sample, and measuring the degree of agglutination of carrier particles as the solid phase. The diagnostic reagent and the method of diagnosis enable many samples to be measured with higher sensitivity and rapidity.

BACKGROUND OF THE INVENTION

The present invention relates to a diagnostic reagent for detectinghepatitis C virus (HCV) infection by utilizing immunoagglutination.

In regard to hepatitis C, HCV gene was detected by the research group ofChiron Corporation, U.S.A., in 1988 prior to the detection of HCV. Todetect antibodies to HCV, various recombinant antigens or syntheticpeptides have been investigated, and kits for detecting HCV-associatedantibodies have been developed. The methods of detection now availableare agar diffusion, counterimmunoelectrophoresis, radioimmunoassay,enzyme immunoassay, passive hemagglutination, and latex agglutination.

Known HCV antigen proteins for use in the detection of HCV-associatedantibodies are core and envelope proteins as structural region proteins,and NS1 to NS5 proteins as non-structural region proteins. One HCVantigen protein alone is not sufficiently high in detection sensitivity,and is also problematical in specificity. Thus, a suitable combinationof proteins in structural and non-structural regions is used (Proc.Natl. Acad. Sci. USA 89:10011-10015, 1992). Attempts to increase thedetection sensitivity further are also made. With the particleagglutination method, for example, the number of HCV antigens forsensitization of particles is increased, or polypeptide having HCVantigenic activity is heat-treated (Japanese Laid-Open PatentPublication No. 1002273/94); alternatively, a fusion protein constructedfrom HCV antigen protein and carrier protein is coated onto hydrophilicparticles for their sensitization (Japanese Laid-Open Patent PublicationNo. 198723/95).

The use of synthetic peptide as an antigen has also been attempted, butthis use is generally said to lower the detection sensitivity.

Thus, there is a growing demand for a diagnostic reagent and a method ofdiagnosis which enable many samples to be measured with high sensitivityand rapidity.

SUMMARY OF THE INVENTION

We have conducted intensive studies in an attempt to attain suchobjectives. As a result, we have found that high sensitivity can berealized by chemically bonding the core antigen, NS3 antigen, NS4antigen or NS5 antigen of HCV to carrier protein by the glutaraldehydemethod to form conjugated antigens, and sensitizing carrier particleswith these conjugated antigens. The use of these conjugated antigens hasalso markedly improved the stability of the particles.

The present invention provides a diagnostic reagent for hepatitis Cvirus infection obtained by sensitizing a solid phase with HCV antigenand a conjugated antigen prepared by chemical bonding of HCV antigen anda carrier protein.

The carrier protein may be any water-soluble protein, preferably the onewith a molecular weight of 10,000 to 1,000,000, more preferably with amolecular weight of 30,000 to 150,000. Preferred examples are bovineserum albumin (BSA), ovalbumin, and hemocyanin. In addition,water-soluble synthetic polymers, such as polyvinyl alcohol and dextran,are also usable.

The solid phase may be carrier particles, a microtiter plate, or a testtube, but carrier particles are preferred. As the carrier particles,known particles generally used in a diagnostic reagent involving theparticle agglutination method can be used. Examples include hydrophobicparticles such as polystyrene latex, copolymer latex particles having ahydrophilic group such as an amino or carboxyl group on the surface ofthe particles, erythrocytes, and gelatin particles. More preferable ispolystyrene latex.

The HCV antigen protein used in the diagnostic reagent of the presentinvention is the known structural region protein or non-structuralregion protein of HCV. The structural region protein may be coreprotein, while the non-structural region protein may be NS3 protein, NS4protein or NS5 protein. The amino acid and nucleotide sequences of theseantigenic proteins are described in the literature (Officially PublishedPatent Gazette No. 508219/93)(SEQ ID NO. 6). Where the antigenic proteinresults from does not matter, so long as it has HCV antigenic activity.Natural isolates, chemical synthetics, and genetic recombinationproducts can be used. Of the proteins in these regions, a peptide ofvarying length can be used as the antigenic protein. Preferably, apeptide composed of 8 or more amino acids containing at least oneepitope is used. More preferably, a synthetic peptide having a molecularweight of 1,000 to 5,000 is used. The peptide can be synthesized by aknown method in the art, such as solid phase synthesis, fragmentcondensation, or classical solution synthesis. Preferably, it can beproduced by the solid phase peptide synthesis method described in theliterature (Merrifield, J. Am. Chem. Soc. 85:2149, 1963). According tothe present invention, one or more of core, NS3, NS4 and NS5 antigenproteins containing one or more different epitopes are combined, and canbe used directly, or after conjugation to a carrier protein, tosensitize carrier particles. In the Examples to be described later on, apeptide containing the 49th to 68th (SEQ ID NO. 6) amino acids in thecore region described in Officially Published Patent Gazette No.508219/93 is used as the core antigen, a peptide containing the 1706thto 1725th (SEQ ID NO. 8), 1718th to 1737th (SEQ ID NO. 9), and 1724th to1743rd (SEQ ID NO. 10) amino acids in the NS4 region described inOfficially Published Patent Gazette No. 508219/93 is used as the NS4peptide, a peptide containing the 2287th to 2306th (SEQ ID NO. 11),2299th to 2318th (SEQ ID NO. 12), and 2311th to 2330th (SEQ ID NO. 13)amino acids in the NS5 region described in Officially Published PatentGazette No. 508219/93 is used as the NS5 peptide, and a peptidecontaining the 1192nd to 1457th (SEQ ID NO. 7) amino acids in the NS3region described in Officially Published Patent Gazette No. 508219/93 isused as the NS3 peptide. However, the antigenic proteins of the presentinvention are not restricted to these peptides.

Each of the above-described antigenic proteins is chemically bonded tothe carrier protein to prepare a conjugated antigen. The antigenicprotein has been found to show higher detection sensitivity whensensitizing the carrier particles as a conjugated antigen than whensensitizing them directly. For the antigen protein with a molecularweight of 10,000 or more like the NS3 antigen used in the presentinvention, however, no marked difference in the sensitivity has beenobserved. Thus, it is permissible to sensitize the carrier particlesdirectly with such a high molecular weight antigen protein, andsensitize the particles with the other antigen proteins as conjugatedantigens. Bonding of the carrier protein and the antigen protein can beperformed by a known method using carbodiimide, periodic acid, maleimideor glutaraldehyde. The use of the glutaraldehyde method is preferred,because their bonding by glutaraldehyde-induced crosslinking increasesreactivity. For the preparation of the conjugated antigen, the carrierprotein and the antigen protein are mixed at a ratio, as the ratio ofthe numbers of molecules for the two, of about 1:3 to 1:20, preferablyabout 1:4 to 1:9, more preferably about 1:6 to 1:8. The so preparedconjugated antigen is bound to(or caused to sensitize) carrier particlesby a known method, which may be, say, physical adsorption or chemicaladsorption. As described previously, the NS3 antigen may be directlycaused to sensitize carrier particles without forming a conjugatedantigen together with the carrier protein. This can be performed by thesame method as described above. Sensitization is carried out in abuffer, a solution with a buffer action, such as phosphate buffer,glycine buffer, TRIS buffer or acetate buffer, preferably at a pH of 3to 8, more preferably at pH 4 to 5.

The present invention also provides a method of diagnosing hepatitis Cvirus infection, which comprises adding the aforementioned diagnosticreagent for hepatitis C virus infection to a sample, and measuring thedegree of agglutination of the carrier particles by a flow cytometer.The diagnostic reagent for hepatitis C virus infection according to thepresent invention also reacts with anti-HCV antibodies, if present inthe sample, to cause agglutination. The resulting agglutination may bemeasured visually or by turbidity or absorbance. However, a rapid,high-sensitivity, high-precision/accuracy measurement can be made byoptically measuring the agglutinated particles with a full-automaticimmunoagglutination measuring system (e.g., PAMIA-30™, TOA MEDICALELECTRONICS Co., Ltd) which relies on the principle of a flow cytometer.In detail, the sample is guided into a flow cell, arranged in a row, andpassed by a sheath flow mechanism. A laser beam is projected onto it,and the intensity of scattered light produced is measured to tell thedegree of agglutination. The number of the agglutinated particles (P:Polymer) and the number of non-agglutinated particles (M: Monomer) arecounted. From the P and M, P/T (T=P+M) is calculated, and the presenceor absence of anti-HCV antibodies is qualitatively evaluated based onthe cutoff value obtained beforehand. This method enables anti-HCVantibodies to be detected with high sensitivity. A suitable particlesize of the particles would make measurement possible by a bloodanalyzer or a particle size analyzer using electrical resistance.However, measurement by an optical method is preferred to avoid aproblem such as clogging of the detector.

PREFERRED EMBODIMENTS OF THE INVENTION

The use of the diagnostic reagent for hepatitis C virus infectionaccording to the present invention permits highly sensitive, earlydiagnosis of infection with hepatitis C virus as compared withcommercially available diagnostic reagents. Actually, the diagnosticreagent of the present invention was tested using panel serum composedof several samples taken over time from the same individual in thecourse of seroconversion of anti-HCV antibodies (e.g., HCVSeroconversion Panel, imported and distributed by Kyowa Medics Co., Ltd,manufactured by BOSTON BIOMEDICA, INC.). The diagnostic reagent forhepatitis C virus infection of the present invention was demonstrated todetect HCV infection in an earlier stage than conventional methods,passive hemagglutination (PHA) using erythrocytes, enzyme immunoassay(EIA) and enzyme-linked immunosorbent assay (ELISA), namely, in theinitial stage of infection.

The diagnostic reagent of the present invention was also compared with adiagnostic reagent produced by using the same antigen as in theinventive diagnostic reagent, but directly causing this antigen tosensitize carrier particles without preparing its conjugated antigentogether with a carrier protein. The diagnostic reagent of the presentinvention was found to be superior in the detection sensitivity.

Furthermore, the diagnostic reagent of the present invention does notdecrease in the detection sensitivity even after long-term storage.Thus, it proves a stable diagnostic reagent.

The diagnostic method of the present invention, compared with aconventional method such as ELISA, does not involve a complicatedwashing step, but can be performed by a mere step of mixing theinventive diagnostic reagent for HCV infection (preferably, latexparticles sensitized with HCV antigen) with a sample (preferably, asubject's blood). The diagnostic method of the present invention alsoenables measurement by a measuring system which performs both of theabove mixing step and the measuring step full-automatically. Thus, thismethod is suitable for measuring many samples.

The present invention will be described in greater detail by referenceto Examples, which do not limit the scope of the invention.

EXAMPLE 1 Preparation of HCV Conjugated Antigens

For use as HCV antigen, NS3 antigen was produced by geneticrecombination based on the description of Example 1 of OfficiallyPublished Patent Gazette No. 508219/93. The NS3 antigen was used the1192nd to 1457th (SEQ ID NO. 7) amino acids of HCV protein.

As core antigen, NS4 antigen and NS5 antigen, peptides containing aminoacid sequences composed of the 49th to 68th (SEQ ID NO. 6), the 1706thto 1725th (SEQ ID NO. 8), and the 2287th to 2306th (SEQ ID NO. 11) aminoacids, respectively, described in Officially Published Patent GazetteNo. 508219/93 were synthesized by the peptide synthesizer Model 431A(PERKIN ELMER).

Seven volumes of a 0.1% (w/v) solution of core antigen (a peptide of the49th to 68th amino acids) in 10 mM PBS, pH 7.0, was added to one volumeof a 0.1% (w/v) solution of BSA (a commercially available product with amolecular weight of 66,000) in 10 mM PBS, pH 7.0. To the mixture, 10 mMPBS, pH 7.0, was further added to make 9 volumes. Then, a 1% aqueoussolution of glutaraldehyde was added to initiate the reaction at areaction temperature of 30° C. Thirty minutes later, one volume of a 20%aqueous solution of glycine was added to terminate the reaction.

A similar procedure was applied to NS3, NS4 and NS5 antigens as well.That is, 1 to 8 volumes of a 0.1% (w/v) solution of the HCV antigen in10 mM PBS, pH 6 to 8, was reacted with 1 volume of a 1% (w/v) solutionof BSA in 10 mM PBS, pH 6 to 8, to prepare HCV conjugated antigens.

EXAMPLE 2 Production of HCV Antigen-sensitized Latex

To a 5% (w/v) dispersion of polystyrene latex particles (SekisuiChemical Co., Ltd) with a particle diameter of 0.78 μm in 10 mM PBS, pH4.0, the NS3 conjugated antigen, the NS4 conjugated antigen and the NS5conjugated antigen prepared in Example 1 was added in an amount of 50 μgeach per ml of the latex dispersion. The mixture was reacted for 24hours at 4° C. Then, the reaction mixture was centrifuged for 10 min at12,000 rpm, and 0.1M PBS, pH 7.0, containing 1 mg/ml BSA was added tothe same concentration as initially added, to disperse the particles.The dispersion was centrifuged again, and dispersed in the same bufferof the same concentration to produce HCV antigen-sensitized latex.

EXAMPLE 3 Agglutination Reaction

To 10 μl of a sample (a subject's blood), 10 μl of the HCVantigen-sensitized latex (5%) prepared in Example 2 was added, and 80 μlof 0.1M phosphate buffer containing 1 mg/ml BSA was further added. Afterthe mixture was reacted for 15 min at 45° C., the degree ofagglutination of the latex particles was measured based onforward-scattered light by means of a full-automatic immunoagglutinationmeasuring system (PAMIA-30™, TOA MEDICAL ELECTRONICS Co., Ltd).

The degree of agglutination is expressed as the percentage of the numberof the agglutinated particles to the total number of the particles (P/T,%).

The results of the measurements are presented in Table 1. As shown inTable 1, a sufficient degree of agglutination was obtained with HCVantibody-positive samples, while no agglutination of latex occurred withHCV antibody-negative samples. Since agglutination was thus observed insamples containing HCV antibodies, one sees that agglutination tookplace upon the reaction of the HCV antigen-sensitized latex particleswith HCV antibodies.

TABLE 1 Degree of agglutination (P/T, %) HCV antibody-positive samplesHCV antibody-negative samples A B C D E F G H I J 72.52 54.56 44.8943.02 73.00 0.95 0.90 0.82 0.82 0.92 Cutoff value: 2.79%

EXAMPLE 4 Detection Sensitivity at Early Stage

The detection sensitivity for HCV antibody seroconversion was testedusing the commercially available HCV Seroconversion Panels PHV901,PHV902 and PHV903 (importer and distributor: Kyowa Medics Co., Ltd,manufacturer: BOSTON BIOMEDICA, INC.) that are composed of severalsamples taken over time from the same individuals in the course ofseroconversion of HCV antibodies. Counting immunoassay (CIA) of thepresent invention using the diagnostic reagent prepared in Example 2 wascompared with the following methods using the products of othercompanies:

Company A: Passive hemagglutination (PHA) using erythrocytes HCVantigens used: Core, NS3, NS4

Company B: Enzyme immunoassay (EIA) HCV antigens used: Core, NS3, NS4

Company C: Enzyme-linked immunosorbent assay (ELISA) HCV antigens used:Core, NS3, NS4

The results obtained are shown in Tables 2, 3 and 4 (the data of CompanyB's and Company C's products in the tables are the values indicated onthe labels attached to the panels). When the diagnostic reagent and thediagnostic method of the present invention were used, HCV infection inPanels PHV902 and PHV903 (the subjects, the presenters of these panels,tested positive for antibodies in PCR from the first day of bloodsampling) was detected earlier than the use of the other companies'products.

TABLE 2 HCV Seroconversion Panel PHV901 Measured value Labeled valuePresent invention Company A Company B Company C Day of Number CIA PHAEIA II ELISA II ID sampling of days Evaluation P/T Evaluation EvaluationCOI* Evaluation COI* PHV901-01 93/09/23  0 − 1.0 − − 0.2 − 0.0 PHV901-0293/11/27  72 − 1.1 − − 0.2 − 0.0 PHV901-03 93/12/29 104 + 73.3 + + 1.6 +1.2 PHV901-04 93/21/31 106 + 74.6 + + 1.7 + 1.2 PHV901-05 94/01/05 111 +75.3 + + 1.7 + 1.4 PHV901-06 94/01/07 113 + 73.8 + + 1.6 + 1.8 PHV901-0794/02/01 138 + 73.8 + + 3.8 + >4 PHV901-08 94/02/09 146 + 71.5 + +3.6 + >4 PHV901-09 93/03/01 166 + 67.0 + + >5 + >4 PHV901-10 94/03/08173 + 63.9 + + >5 + >4 PHV901-11 94/04/14 209 + 61.7 + + >5 + >4 Cutoff:1.75% COI* = Cutoff index

TABLE 3 HCV Seroconversion Panel PHV902 Measured value Labeled valuePresent invention Company A Company B Company C Day of Number CIA PHAEIA II ELISA II ID sampling of days Evaluation P/T Evaluation EvaluationCOI* Evaluation COI* PCR PHV902-01 92/02/07  0 − 1.0 − − 0.2 − 0.2 +PHV902-02 92/02/12  2 − 1.0 − − 0.2 − 0.2 + PHV902-03 92/02/17  7 + 3.0− − 0.4 − 0.3 + PHV902-04 92/02/19  9 + 4.4 + − 0.8 − 0.5 + PHV902-0592/02/24 14 + 17.0 + + 3.9 + >4 + PHV902-06 92/02/26 16 + 18.0 + +5.0 + >4 + PHV902-07 92/03/02 21 + 17.6 + + >5 + >4 + Cutoff: 1.75% COI*= Cutoff index

TABLE 4 HCV Seroconversion Panel PHV903 Measured value Labeled valuePresent invention Company A Company B Company C Day of Number CIA PHAEIA II ELISA II ID sampling of days Evaluation P/T Evaluation EvaluationCOI* Evaluation COI* PCR PHV903-01 92/02/07  0 − 0.8 − − 0.2 − 0.2 +PHV903-02 92/02/12  5 + 2.2 − − 0.4 − 0.6 + PHV903-03 92/02/14  7 + 4.2− − 0.5 − 0.7 + PHV903-04 92/02/19 12 + 12.9 + − 0.8 + 1.5 + PHV903-0592/02/21 14 + 11.5 + − 0.7 + 1.5 + PHV903-06 92/02/26 19 + 28.2 + +1.7 + 4.0 + PHV903-07 92/02/28 21 + 29.1 + + 1.9 + >4 + PHV903-0892/03/04 26 + 26.6 + + 2.3 + >4 + Cutoff: 1.75% COI* = Cutoff index

Table 5 Comparison with Diagnostic Reagent Prepared by DirectSensitization with HCV Antigen

The diagnostic reagent of the present invention and a diagnostic reagentprepared by direct sensitization with the same HCV antigen as in theinventive diagnostic reagent were tested for sensitivity for detectionof HCV antibodies.

NS3 antigen (obtained by genetic recombination), core antigen (peptideobtained by synthesis), NS4 antigen (peptide obtained by synthesis) andNS5 antigen (peptide obtained by synthesis) were converted intoconjugated antigens together with BSA, and used for sensitization in thesame manner as in Example 2. Thus, HCV conjugated antigen-sensitizedlatex was produced.

As a control, HCV antigen-directly-sensitized latex was produced bydirect sensitization using the same antigens and the same conditions forsensitization.

Using these latices, the degree of agglutination (P/T, %) of HCVantibody-positive samples (A to E) and that of HCV antibody-negativesamples (F to J) were measured by the method of Example 3. Whether eachsample was positive or negative for HCV antibodies was evaluated in viewof the cutoff value (1.95% for the conjugated antigen-sensitized latex;1.01% for the antigen-directly-sensitized latex). The results are shownin Table 5.

TABLE 5 Conjugated antigen-sensitized latex Antigen-directly-sensitizedlatex Sample Degree of agglutination (%) Evaluation Degree ofagglutination (%) Evaluation A 15.87 Positive 0.34 Negative B 66.28Positive 24.36 Positive C 35.28 Positive 6.35 Positive D 50.98 Positive15.56 Positive E 17.88 Positive 0.55 Negative F 0.46 Negative 0.34Negative G 0.56 Negative 0.36 Negative H 0.79 Negative 0.35 Negative I0.62 Negative 0.56 Negative J 0.65 Negative 0.55 Negative

In the HCV antibody-positive samples A and E, HCV antibodies were notdetected with the diagnostic reagents involving direct sensitizationwith the antigens, but were detected with the diagnostic reagent of thepresent invention involving sensitization with the conjugated antigens.

EXAMPLE 6 Test for Long-term Storage Stability

The 5% (w/v) suspensions of HCV antigen-sensitized latices in 0.1M PBS,pH 7.0, in Example 5 were stored in a refrigerated condition to examinethe storage stability of the diagnostic reagents. The results are shownin Table 6.

TABLE 6 HCV antibody-negative pooled serum HCV antibody-positive pooledserum Cutoff value Conjugated antigen- Conjugated antigen- Conjugatedantigen- sensitized Directly sensitized sensitized Directly sensitizedsensitized Directly sensitized  0 month 0.71% 0.75% 41.90% 12.46% 2.35%1.25%  1 month 0.71% 0.72% 40.85% 11.56% 2.51% 1.35%  3 months 0.75%1.02% 46.83% 10.79% 2.56% 1.56%  6 months 0.78% 1.22% 43.47% 10.62%2.55% 1.82%  9 months 0.82% 1.50% 43.86% 9.79% 2.25% 2.10% 12 months0.75% 1.62% 41.62% 8.62% 2.33% 2.32% 13 months 0.78% 1.97% 43.35% 8.65%2.44% 2.57%

The above results demonstrate that the conjugated antigen-sensitizedlatex was stable in terms of the degree of agglutination even whenstored for a long period of 13 months. In the case of the directlysensitized latex, on the other hand, the test using HCVantibody-negative pooled serum showed gradual increases in the degree ofagglutination during long-term storage, while the test using HCVantibody-positive pooled serum showed gradual decreases in the degree ofagglutination. Gradual increases in the cutoff value were also observedwith the directly sensitized latex.

EXAMPLE 7 Production (2) of HCV Antigen-sensitized Latex

HCV antigen-sensitized latex was prepared by the same procedure as inExample 2 with the use of the same HCV antigens as in Example 1, exceptthat NS3 antigen was not formed into conjugated antigen, but was usedfor direct sensitization.

EXAMPLE 8 Production (3) of HCV Antigen-sensitized Latex

The same NS3 antigen as in Example 1 was used. Core antigen was apeptide of the 49th to 68th (SEQ ID NO. 6) amino acids described in theaforementioned publication (Officially Published Patent Gazette No.508219/93). NS4 antigen was peptides of the 1706th to 1725th (SEQ ID NO.8) and the 1718th (SEQ ID NO. 9) to 1737th amino acids described there.NS5 antigen was peptides of the 2287th to 2306th (SEQ ID NO. 11) and the2299th to 2318th (SEQ ID NO. 12) amino acids described there. As inExample 1, 1 to 8 volumes of a 0.1% (w/v) HCV antigen solution wasreacted with 1 volume of a 1% (w/v) BSA solution to prepare HCVconjugated antigens. Using them, HCV antigen-sensitized latex wasprepared in the same manner as in Example 2.

EXAMPLE 9 Production (4) of HCV Antigen-sensitized Latex

The same NS3 antigen as in Example 1 was used. Core antigen was apeptide of the 49th to 68th (SEQ ID NO. 6) amino acids described in theaforementioned publication. NS4 antigen was peptides of the 1706th to1725th (SEQ ID NO. 8), the 1718th to 1737th (SEQ ID NO. 9) and the1724th to 1743rd (SEQ ID NO. 10) amino acids described there. NS5antigen was peptides of the 2287th to 2306th (SEQ ID NO. 11), the 2299thto 2318th (SEQ ID NO. 12) and the 2311th to 2330th (SEQ ID NO. 13) aminoacids described there. As in Example 1, 1 to 8 volumes of a 0.1% (w/v)HCV antigen solution was reacted with 1 volume of a 1% (w/v) BSAsolution to prepare HCV conjugated antigens. Using them, HCVantigen-sensitized latex was prepared in the same manner as in Example2.

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410 415 Asn Gly Ser Trp His Leu Asn Ser Thr Ala Leu Asn Cys AsnAsp Ser 420 425 430 Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr His HisLys Phe Asn 435 440 445 Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys ArgPro Leu Thr Asp 450 455 460 Phe Asp Gln Gly Trp Gly Pro Ile Ser Tyr AlaAsn Gly Ser Gly Pro 465 470 475 480 Asp Gln Arg Pro Tyr Cys Trp His TyrPro Pro Lys Pro Cys Gly Ile 485 490 495 Val Pro Ala Lys Ser Val Cys GlyPro Val Tyr Cys Phe Thr Pro Ser 500 505 510 Pro Val Val Val Gly Thr ThrAsp Arg Ser Gly Ala Pro Thr Tyr Ser 515 520 525 Trp Gly Glu Asn Asp ThrAsp Val Phe Val Leu Asn Asn Thr Arg Pro 530 535 540 Pro Leu Gly Asn TrpPhe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe 545 550 555 560 Thr Lys ValCys Gly Ala Pro Pro Cys Val Ile Gly Gly Ala Gly Asn 565 570 575 Asn ThrLeu His Cys Pro Thr Asp Cys Phe Arg Lys His Pro Asp Ala 580 585 590 ThrTyr Ser Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Leu 595 600 605Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile Asn Tyr 610 615620 Thr Ile Phe Lys Ile Arg Met Tyr Val Gly Gly Val Glu His Arg Leu 625630 635 640 Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu GluAsp 645 650 655 Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr ThrGln Trp 660 665 670 Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala LeuSer Thr Gly 675 680 685 Leu Ile His Leu His Gln Asn Ile Val Asp Val GlnTyr Leu Tyr Gly 690 695 700 Val Gly Ser Ser Ile Ala Ser Trp Ala Ile LysTrp Glu Tyr Val Val 705 710 715 720 Leu Leu Phe Leu Leu Leu Ala Asp AlaArg Val Cys Ser Cys Leu Trp 725 730 735 Met Met Leu Leu Ile Ser Gln AlaGlu Ala Ala Leu Glu Asn Leu Val 740 745 750 Ile Leu Asn Ala Ala Ser LeuAla Gly Thr His Gly Leu Val Ser Phe 755 760 765 Leu Val Phe Phe Cys PheAla Trp Tyr Leu Lys Gly Lys Trp Val Pro 770 775 780 Gly Ala Val Tyr ThrPhe Tyr Gly Met Trp Pro Leu Leu Leu Leu Leu 785 790 795 800 Leu Ala LeuPro Gln Arg Ala Tyr Ala Leu Asp Thr Glu Val Ala Ala 805 810 815 Ser CysGly Gly Val Val Leu Val Gly Leu Met Ala Leu Thr Leu Ser 820 825 830 ProTyr Tyr Lys Arg Tyr Ile Ser Trp Cys Leu Trp Trp Leu Gln Tyr 835 840 845Phe Leu Thr Arg Val Glu Ala Gln Leu His Val Trp Ile Pro Pro Leu 850 855860 Asn Val Arg Gly Gly Arg Asp Ala Val Ile Leu Leu Met Cys Ala Val 865870 875 880 His Pro Thr Leu Val Phe Asp Ile Thr Lys Leu Leu Leu Ala ValPhe 885 890 895 Gly Pro Leu Trp Ile Leu Gln Ala Ser Leu Leu Lys Val ProTyr Phe 900 905 910 Val Arg Val Gln Gly Leu Leu Arg Phe Cys Ala Leu AlaArg Lys Met 915 920 925 Ile Gly Gly His Tyr Val Gln Met Val Ile Ile LysLeu Gly Ala Leu 930 935 940 Thr Gly Thr Tyr Val Tyr Asn His Leu Thr ProLeu Arg Asp Trp Ala 945 950 955 960 His Asn Gly Leu Arg Asp Leu Ala ValAla Val Glu Pro Val Val Phe 965 970 975 Ser Gln Met Glu Thr Lys Leu IleThr Trp Gly Ala Asp Thr Ala Ala 980 985 990 Cys Gly Asp Ile Ile Asn GlyLeu Pro Val Ser Ala Arg Arg Gly Arg 995 1000 1005 Glu Ile Leu Leu GlyPro Ala Asp Gly Met Val Ser Lys Gly Trp Arg 1010 1015 1020 Leu Leu AlaPro Ile Thr Ala Tyr Ala Gln Gln Thr Arg Gly Leu Leu 1025 1030 1035 104Gly Cys Ile Ile Thr Ser Leu Thr Gly Arg Asp Lys Asn Gln Val Glu 10451050 1055 Gly Glu Val Gln Ile Val Ser Thr Ala Ala Gln Thr Phe Leu AlaThr 1060 1065 1070 Cys Ile Asn Gly Val Cys Trp Thr Val Tyr His Gly AlaGly Thr Arg 1075 1080 1085 Thr Ile Ala Ser Pro Lys Gly Pro Val Ile GlnMet Tyr Thr Asn Val 1090 1095 1100 Asp Gln Asp Leu Val Gly Trp Pro AlaPro Gln Gly Ser Arg Ser Leu 1105 1110 1115 112 Thr Pro Cys Thr Cys GlySer Ser Asp Leu Tyr Leu Val Thr Arg His 1125 1130 1135 Ala Asp Val IlePro Val Arg Arg Arg Gly Asp Ser Arg Gly Ser Leu 1140 1145 1150 Leu SerPro Arg Pro Ile Ser Tyr Leu Lys Gly Ser Ser Gly Gly Pro 1155 1160 1165Leu Leu Cys Pro Ala Gly His Ala Val Gly Ile Phe Arg Ala Ala Val 11701175 1180 Cys Thr Arg Gly Val Ala Lys Ala Val Asp Phe Ile Pro Val GluAsn 1185 1190 1195 120 Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr AspAsn Ser Ser Pro 1205 1210 1215 Pro Val Val Pro Gln Ser Phe Gln Val AlaHis Leu His Ala Pro Thr 1220 1225 1230 Gly Ser Gly Lys Ser Thr Lys ValPro Ala Ala Tyr Ala Ala Gln Gly 1235 1240 1245 Tyr Lys Val Leu Val LeuAsn Pro Ser Val Ala Ala Thr Leu Gly Phe 1250 1255 1260 Gly Ala Tyr MetSer Lys Ala His Gly Ile Asp Pro Asn Ile Arg Thr 1265 1270 1275 128 GlyVal Arg Thr Ile Thr Thr Gly Ser Pro Ile Thr Tyr Ser Thr Tyr 1285 12901295 Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly Gly Ala Tyr Asp Ile1300 1305 1310 Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala Thr Ser IleLeu Gly 1315 1320 1325 Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala GlyAla Arg Leu Val 1330 1335 1340 Val Leu Ala Thr Ala Thr Pro Pro Gly SerVal Thr Val Pro His Pro 1345 1350 1355 136 Asn Ile Glu Glu Val Ala LeuSer Thr Thr Gly Glu Ile Pro Phe Tyr 1365 1370 1375 Gly Lys Ala Ile ProLeu Glu Val Ile Lys Gly Gly Arg His Leu Ile 1380 1385 1390 Phe Cys HisSer Lys Lys Lys Cys Asp Glu Leu Ala Ala Lys Leu Val 1395 1400 1405 AlaLeu Gly Ile Asn Ala Val Ala Tyr Tyr Arg Gly Leu Asp Val Ser 1410 14151420 Val Ile Pro Thr Ser Gly Asp Val Val Val Val Ala Thr Asp Ala Leu1425 1430 1435 144 Met Thr Gly Tyr Thr Gly Asp Phe Asp Ser Val Ile AspCys Asn Thr 1445 1450 1455 Cys Val Thr Gln Thr Val Asp Phe Ser Leu AspPro Thr Phe Thr Ile 1460 1465 1470 Glu Thr Ile Thr Leu Pro Gln Asp AlaVal Ser Arg Thr Gln Arg Arg 1475 1480 1485 Gly Arg Thr Gly Arg Gly LysPro Gly Ile Tyr Arg Phe Val Ala Pro 1490 1495 1500 Gly Glu Arg Pro SerGly Met Phe Asp Ser Ser Val Leu Cys Glu Cys 1505 1510 1515 152 Tyr AspAla Gly Cys Ala Trp Tyr Glu Leu Thr Pro Ala Glu Thr Thr 1525 1530 1535Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly Leu Pro Val Cys Gln 15401545 1550 Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr Gly Leu Thr HisIle 1555 1560 1565 Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser Gly GluAsn Leu Pro 1570 1575 1580 Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys AlaArg Ala Gln Ala Pro 1585 1590 1595 160 Pro Pro Ser Trp Asp Gln Met TrpLys Cys Leu Ile Arg Leu Lys Pro 1605 1610 1615 Thr Leu His Gly Pro ThrPro Leu Leu Tyr Arg Leu Gly Ala Val Gln 1620 1625 1630 Asn Glu Ile ThrLeu Thr His Pro Val Thr Lys Tyr Ile Met Thr Cys 1635 1640 1645 Met SerAla Asp Leu Glu Val Val Thr Ser Thr Trp Val Leu Val Gly 1650 1655 1660Gly Val Leu Ala Ala Leu Ala Ala Tyr Cys Leu Ser Thr Gly Cys Val 16651670 1675 168 Val Ile Val Gly Arg Val Val Leu Ser Gly Lys Pro Ala IleIle Pro 1685 1690 1695 Asp Arg Glu Val Leu Tyr Arg Glu Phe Asp Glu MetGlu Glu Cys Ser 1700 1705 1710 Gln His Leu Pro Tyr Ile Glu Gln Gly MetMet Leu Ala Glu Gln Phe 1715 1720 1725 Lys Gln Lys Ala Leu Gly Leu LeuGln Thr Ala Ser Arg Gln Ala Glu 1730 1735 1740 Val Ile Ala Pro Ala ValGln Thr Asn Trp Gln Lys Leu Glu Thr Phe 1745 1750 1755 176 Trp Ala LysHis Met Trp Asn Phe Ile Ser Gly Ile Gln Tyr Leu Ala 1765 1770 1775 GlyLeu Ser Thr Leu Pro Gly Asn Pro Ala Ile Ala Ser Leu Met Ala 1780 17851790 Phe Thr Ala Ala Val Thr Ser Pro Leu Thr Thr Ser Gln Thr Leu Leu1795 1800 1805 Phe Asn Ile Leu Gly Gly Trp Val Ala Ala Gln Leu Ala AlaPro Gly 1810 1815 1820 Ala Ala Thr Ala Phe Val Gly Ala Gly Leu Ala GlyAla Ala Ile Gly 1825 1830 1835 184 Ser Val Gly Leu Gly Lys Val Leu IleAsp Ile Leu Ala Gly Tyr Gly 1845 1850 1855 Ala Gly Val Ala Gly Ala LeuVal Ala Phe Lys Ile Met Ser Gly Glu 1860 1865 1870 Val Pro Ser Thr GluAsp Leu Val Asn Leu Leu Pro Ala Ile Leu Ser 1875 1880 1885 Pro Gly AlaLeu Val Val Gly Val Val Cys Ala Ala Ile Leu Arg Arg 1890 1895 1900 HisVal Gly Pro Gly Glu Gly Ala Val Gln Trp Met Asn Arg Leu Ile 1905 19101915 192 Ala Phe Ala Ser Arg Gly Asn His Val Ser Pro Thr His Tyr Val Pro1925 1930 1935 Glu Ser Asp Ala Ala Ala Arg Val Thr Ala Ile Leu Ser SerLeu Thr 1940 1945 1950 Val Thr Gln Leu Leu Arg Arg Leu His Gln Trp IleSer Ser Glu Cys 1955 1960 1965 Thr Thr Pro Cys Ser Gly Ser Trp Leu ArgAsp Ile Trp Asp Trp Ile 1970 1975 1980 Cys Glu Val Leu Ser Asp Phe LysThr Trp Leu Lys Ala Lys Leu Met 1985 1990 1995 200 Pro Gln Leu Pro GlyIle Pro Phe Val Ser Cys Gln Arg Gly Tyr Lys 2005 2010 2015 Gly Val TrpArg Gly Asp Gly Ile Met His Thr Arg Cys His Cys Gly 2020 2025 2030 AlaGlu Ile Thr Gly His Val Lys Asn Gly Thr Met Arg Ile Val Gly 2035 20402045 Pro Arg Thr Cys Arg Asn Met Trp Ser Gly Thr Phe Pro Ile Asn Ala2050 2055 2060 Tyr Thr Thr Gly Pro Cys Thr Pro Leu Pro Ala Pro Asn TyrThr Phe 2065 2070 2075 208 Ala Leu Trp Arg Val Ser Ala Glu Glu Tyr ValGlu Ile Arg Gln Val 2085 2090 2095 Gly Asp Phe His Tyr Val Thr Gly MetThr Thr Asp Asn Leu Lys Cys 2100 2105 2110 Pro Cys Gln Val Pro Ser ProGlu Phe Phe Thr Glu Leu Asp Gly Val 2115 2120 2125 Arg Leu His Arg PheAla Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu 2130 2135 2140 Val Ser PheArg Val Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu 2145 2150 2155 216Pro Cys Glu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr 21652170 2175 Asp Pro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu AlaArg 2180 2185 2190 Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Ala Ser GlnLeu Ser Ala 2195 2200 2205 Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn HisAsp Ser Pro Asp Ala 2210 2215 2220 Glu Leu Ile Glu Ala Asn Leu Leu TrpArg Gln Glu Met Gly Gly Asn 2225 2230 2235 224 Ile Thr Arg Val Glu SerGlu Asn Lys Val Val Ile Leu Asp Ser Phe 2245 2250 2255 Asp Pro Leu ValAla Glu Glu Asp Glu Arg Glu Ile Ser Val Pro Ala 2260 2265 2270 Glu IleLeu Arg Lys Ser Arg Arg Phe Ala Gln Ala Leu Pro Val Trp 2275 2280 2285Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val Glu Thr Trp Lys Lys Pro 22902295 2300 Asp Tyr Glu Pro Pro Val Val His Gly Cys Pro Leu Pro Pro ProLys 2305 2310 2315 232 Ser Pro Pro Val Pro Pro Pro Arg Lys Lys Arg ThrVal Val Leu Thr 2325 2330 2335 Glu Ser Thr Leu Ser Thr Ala Leu Ala GluLeu Ala Thr Arg Ser Phe 2340 2345 2350 Gly Ser Ser Ser Thr Ser Gly IleThr Gly Asp Asn Thr Thr Thr Ser 2355 2360 2365 Ser Glu Pro Ala Pro SerGly Cys Pro Pro Asp Ser Asp Ala Glu Ser 2370 2375 2380 Tyr Ser Ser MetPro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu 2385 2390 2395 240 SerAsp Gly Ser Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp 2405 24102415 Val Val Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr2420 2425 2430 Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn Ala LeuSer Asn 2435 2440 2445 Ser Leu Leu Arg His His Asn Leu Val Tyr Ser ThrThr Ser Arg Ser 2450 2455 2460 Ala Cys Gln Arg Gln Lys Lys Val Thr PheAsp Arg Leu Gln Val Leu 2465 2470 2475 248 Asp Ser His Tyr Gln Asp ValLeu Lys Glu Val Lys Ala Ala Ala Ser 2485 2490 2495 Lys Val Lys Ala AsnLeu Leu Ser Val Glu Glu Ala Cys Ser Leu Thr 2500 2505 2510 Pro Pro HisSer Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val 2515 2520 2525 ArgCys His Ala Arg Lys Ala Val Thr His Ile Asn Ser Val Trp Lys 2530 25352540 Asp Leu Leu Glu Asp Asn Val Thr Pro Ile Asp Thr Thr Ile Met Ala2545 2550 2555 256 Lys Asn Glu Val Phe Cys Val Gln Pro Glu Lys Gly GlyArg Lys Pro 2565 2570 2575 Ala Arg Leu Ile Val Phe Pro Asp Leu Gly ValArg Val Cys Glu Lys 2580 2585 2590 Met Ala Leu Tyr Asp Val Val Thr LysLeu Pro Leu Ala Val Met Gly 2595 2600 2605 Ser Ser Tyr Gly Phe Gln TyrSer Pro Gly Gln Arg Val Glu Phe Leu 2610 2615 2620 Val Gln Ala Trp LysSer Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp 2625 2630 2635 264 Thr ArgCys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu 2645 2650 2655Glu Ala Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala 26602665 2670 Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu ThrAsn 2675 2680 2685 Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg AlaSer Gly Val 2690 2695 2700 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr CysTyr Ile Lys Ala Arg 2705 2710 2715 272 Ala Ala Cys Arg Ala Ala Gly LeuGln Asp Cys Thr Met Leu Val Cys 2725 2730 2735 Gly Asp Asp Leu Val ValIle Cys Glu Ser Ala Gly Val Gln Glu Asp 2740 2745 2750 Ala Ala Ser LeuArg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala 2755 2760 2765 Pro ProGly Asp Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr 2770 2775 2780Ser Cys Ser Ser Asn Val Ser Val Ala His Asp Gly Ala Gly Lys Arg 27852790 2795 280 Val Tyr Tyr Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala ArgAla Ala 2805 2810 2815 Trp Glu Thr Ala Arg His Thr Pro Val Asn Ser TrpLeu Gly Asn Ile 2820 2825 2830 Ile Met Phe Ala Pro Thr Leu Trp Ala ArgMet Ile Leu Met Thr His 2835 2840 2845 Phe Phe Ser Val Leu Ile Ala ArgAsp Gln Leu Glu Gln Ala Leu Asp 2850 2855 2860 Cys Glu Ile Tyr Gly AlaCys Tyr Ser Ile Glu Pro Leu Asp Leu Pro 2865 2870 2875 288 Pro Ile IleGln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser 2885 2890 2895 TyrSer Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu 2900 29052910 Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg2915 2920 2925 Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala Ala Ile Cys GlyLys Tyr 2930 2935 2940 Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys LeuThr Pro Ile Ala 2945 2950 2955 296 Ala Ala Gly Gln Leu Asp Leu Ser GlyTrp Phe Thr Ala Gly Tyr Ser 2965 2970 2975 Gly Gly Asp Ile Tyr His SerVal Ser His Ala Arg Pro Arg Trp Ile 2980 2985 2990 Trp Phe Cys Leu LeuLeu Leu Ala Ala Gly Val Gly Ile Tyr Leu Leu 2995 3000 3005 Pro Asn Arg3010 120 amino acids amino acid single linear peptide 2 Met Ser Thr AsnPro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn 1 5 10 15 Arg Arg ProGln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 Gly Val TyrLeu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 Thr Arg LysThr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 Ile Pro LysAla Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly 65 70 75 80 Tyr ProTrp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp 85 90 95 Leu LeuSer Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 ArgArg Arg Ser Arg Asn Leu Gly 115 120 590 amino acids amino acid singlelinear peptide 3 Asp Lys Asn Gln Val Glu Gly Glu Val Gln Ile Val Ser ThrAla Ala 1 5 10 15 Gln Thr Phe Leu Ala Thr Cys Ile Asn Gly Val Cys TrpThr Val Tyr 20 25 30 His Gly Ala Gly Thr Arg Thr Ile Ala Ser Pro Lys GlyPro Val Ile 35 40 45 Gln Met Tyr Thr Asn Val Asp Gln Asp Leu Val Gly TrpPro Ala Pro 50 55 60 Gln Gly Ser Arg Ser Leu Thr Pro Cys Thr Cys Gly SerSer Asp Leu 65 70 75 80 Tyr Leu Val Thr Arg His Ala Asp Val Ile Pro ValArg Arg Arg Gly 85 90 95 Asp Ser Arg Gly Ser Leu Leu Ser Pro Arg Pro IleSer Tyr Leu Lys 100 105 110 Gly Ser Ser Gly Gly Pro Leu Leu Cys Pro AlaGly His Ala Val Gly 115 120 125 Ile Phe Arg Ala Ala Val Cys Thr Arg GlyVal Ala Lys Ala Val Asp 130 135 140 Phe Ile Pro Val Glu Asn Leu Glu ThrThr Met Arg Ser Pro Val Phe 145 150 155 160 Thr Asp Asn Ser Ser Pro ProVal Val Pro Gln Ser Phe Gln Val Ala 165 170 175 His Leu His Ala Pro ThrGly Ser Gly Lys Ser Thr Lys Val Pro Ala 180 185 190 Ala Tyr Ala Ala GlnGly Tyr Lys Val Leu Val Leu Asn Pro Ser Val 195 200 205 Ala Ala Thr LeuGly Phe Gly Ala Tyr Met Ser Lys Ala His Gly Ile 210 215 220 Asp Pro AsnIle Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser Pro 225 230 235 240 IleThr Tyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys Ser 245 250 255Gly Gly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp 260 265270 Ala Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr 275280 285 Ala Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser290 295 300 Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser ThrThr 305 310 315 320 Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu GluVal Ile Lys 325 330 335 Gly Gly Arg His Leu Ile Phe Cys His Ser Lys LysLys Cys Asp Glu 340 345 350 Leu Ala Ala Lys Leu Val Ala Leu Gly Ile AsnAla Val Ala Tyr Tyr 355 360 365 Arg Gly Leu Asp Val Ser Val Ile Pro ThrSer Gly Asp Val Val Val 370 375 380 Val Ala Thr Asp Ala Leu Met Thr GlyTyr Thr Gly Asp Phe Asp Ser 385 390 395 400 Val Ile Asp Cys Asn Thr CysVal Thr Gln Thr Val Asp Phe Ser Leu 405 410 415 Asp Pro Thr Phe Thr IleGlu Thr Ile Thr Leu Pro Gln Asp Ala Val 420 425 430 Ser Arg Thr Gln ArgArg Gly Arg Thr Gly Arg Gly Lys Pro Gly Ile 435 440 445 Tyr Arg Phe ValAla Pro Gly Glu Arg Pro Ser Gly Met Phe Asp Ser 450 455 460 Ser Val LeuCys Glu Cys Tyr Asp Ala Gly Cys Ala Trp Tyr Glu Leu 465 470 475 480 ThrPro Ala Glu Thr Thr Val Arg Leu Arg Ala Tyr Met Asn Thr Pro 485 490 495Gly Leu Pro Val Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe 500 505510 Thr Gly Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln 515520 525 Ser Gly Glu Asn Leu Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys530 535 540 Ala Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met Trp LysCys 545 550 555 560 Leu Ile Arg Leu Lys Pro Thr Leu His Gly Pro Thr ProLeu Leu Tyr 565 570 575 Arg Leu Gly Ala Val Gln Asn Glu Ile Thr Leu ThrHis Pro 580 585 590 360 amino acids amino acid single linear peptide 4Val Thr Lys Tyr Ile Met Thr Cys Met Ser Ala Asp Leu Glu Val Val 1 5 1015 Thr Ser Thr Trp Val Leu Val Gly Gly Val Leu Ala Ala Leu Ala Ala 20 2530 Tyr Cys Leu Ser Thr Gly Cys Val Val Ile Val Gly Arg Val Val Leu 35 4045 Ser Gly Lys Pro Ala Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu 50 5560 Phe Asp Glu Met Glu Glu Cys Ser Gln His Leu Pro Tyr Ile Glu Gln 65 7075 80 Gly Met Met Leu Ala Glu Gln Phe Lys Gln Lys Ala Leu Gly Leu Leu 8590 95 Gln Thr Ala Ser Arg Gln Ala Glu Val Ile Ala Pro Ala Val Gln Thr100 105 110 Asn Trp Gln Lys Leu Glu Thr Phe Trp Ala Lys His Met Trp AsnPhe 115 120 125 Ile Ser Gly Ile Gln Tyr Leu Ala Gly Leu Ser Thr Leu ProGly Asn 130 135 140 Pro Ala Ile Ala Ser Leu Met Ala Phe Thr Ala Ala ValThr Ser Pro 145 150 155 160 Leu Thr Thr Ser Gln Thr Leu Leu Phe Asn IleLeu Gly Gly Trp Val 165 170 175 Ala Ala Gln Leu Ala Ala Pro Gly Ala AlaThr Ala Phe Val Gly Ala 180 185 190 Gly Leu Ala Gly Ala Ala Ile Gly SerVal Gly Leu Gly Lys Val Leu 195 200 205 Ile Asp Ile Leu Ala Gly Tyr GlyAla Gly Val Ala Gly Ala Leu Val 210 215 220 Ala Phe Lys Ile Met Ser GlyGlu Val Pro Ser Thr Glu Asp Leu Val 225 230 235 240 Asn Leu Leu Pro AlaIle Leu Ser Pro Gly Ala Leu Val Val Gly Val 245 250 255 Val Cys Ala AlaIle Leu Arg Arg His Val Gly Pro Gly Glu Gly Ala 260 265 270 Val Gln TrpMet Asn Arg Leu Ile Ala Phe Ala Ser Arg Gly Asn His 275 280 285 Val SerPro Thr His Tyr Val Pro Glu Ser Asp Ala Ala Ala Arg Val 290 295 300 ThrAla Ile Leu Ser Ser Leu Thr Val Thr Gln Leu Leu Arg Arg Leu 305 310 315320 His Gln Trp Ile Ser Ser Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp 325330 335 Leu Arg Asp Ile Trp Asp Trp Ile Cys Glu Val Leu Ser Asp Phe Lys340 345 350 Thr Trp Leu Lys Ala Lys Leu Met 355 360 1011 amino acidsamino acid single linear peptide 5 Pro Gln Leu Pro Gly Ile Pro Phe ValSer Cys Gln Arg Gly Tyr Lys 1 5 10 15 Gly Val Trp Arg Gly Asp Gly IleMet His Thr Arg Cys His Cys Gly 20 25 30 Ala Glu Ile Thr Gly His Val LysAsn Gly Thr Met Arg Ile Val Gly 35 40 45 Pro Arg Thr Cys Arg Asn Met TrpSer Gly Thr Phe Pro Ile Asn Ala 50 55 60 Tyr Thr Thr Gly Pro Cys Thr ProLeu Pro Ala Pro Asn Tyr Thr Phe 65 70 75 80 Ala Leu Trp Arg Val Ser AlaGlu Glu Tyr Val Glu Ile Arg Gln Val 85 90 95 Gly Asp Phe His Tyr Val ThrGly Met Thr Thr Asp Asn Leu Lys Cys 100 105 110 Pro Cys Gln Val Pro SerPro Glu Phe Phe Thr Glu Leu Asp Gly Val 115 120 125 Arg Leu His Arg PheAla Pro Pro Cys Lys Pro Leu Leu Arg Glu Glu 130 135 140 Val Ser Phe ArgVal Gly Leu His Glu Tyr Pro Val Gly Ser Gln Leu 145 150 155 160 Pro CysGlu Pro Glu Pro Asp Val Ala Val Leu Thr Ser Met Leu Thr 165 170 175 AspPro Ser His Ile Thr Ala Glu Ala Ala Gly Arg Arg Leu Ala Arg 180 185 190Gly Ser Pro Pro Ser Val Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala 195 200205 Pro Ser Leu Lys Ala Thr Cys Thr Ala Asn His Asp Ser Pro Asp Ala 210215 220 Glu Leu Ile Glu Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn225 230 235 240 Ile Thr Arg Val Glu Ser Glu Asn Lys Val Val Ile Leu AspSer Phe 245 250 255 Asp Pro Leu Val Ala Glu Glu Asp Glu Arg Glu Ile SerVal Pro Ala 260 265 270 Glu Ile Leu Arg Lys Ser Arg Arg Phe Ala Gln AlaLeu Pro Val Trp 275 280 285 Ala Arg Pro Asp Tyr Asn Pro Pro Leu Val GluThr Trp Lys Lys Pro 290 295 300 Asp Tyr Glu Pro Pro Val Val His Gly CysPro Leu Pro Pro Pro Lys 305 310 315 320 Ser Pro Pro Val Pro Pro Pro ArgLys Lys Arg Thr Val Val Leu Thr 325 330 335 Glu Ser Thr Leu Ser Thr AlaLeu Ala Glu Leu Ala Thr Arg Ser Phe 340 345 350 Gly Ser Ser Ser Thr SerGly Ile Thr Gly Asp Asn Thr Thr Thr Ser 355 360 365 Ser Glu Pro Ala ProSer Gly Cys Pro Pro Asp Ser Asp Ala Glu Ser 370 375 380 Tyr Ser Ser MetPro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu 385 390 395 400 Ser AspGly Ser Trp Ser Thr Val Ser Ser Glu Ala Asn Ala Glu Asp 405 410 415 ValVal Cys Cys Ser Met Ser Tyr Ser Trp Thr Gly Ala Leu Val Thr 420 425 430Pro Cys Ala Ala Glu Glu Gln Lys Leu Pro Ile Asn Ala Leu Ser Asn 435 440445 Ser Leu Leu Arg His His Asn Leu Val Tyr Ser Thr Thr Ser Arg Ser 450455 460 Ala Cys Gln Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu465 470 475 480 Asp Ser His Tyr Gln Asp Val Leu Lys Glu Val Lys Ala AlaAla Ser 485 490 495 Lys Val Lys Ala Asn Leu Leu Ser Val Glu Glu Ala CysSer Leu Thr 500 505 510 Pro Pro His Ser Ala Lys Ser Lys Phe Gly Tyr GlyAla Lys Asp Val 515 520 525 Arg Cys His Ala Arg Lys Ala Val Thr His IleAsn Ser Val Trp Lys 530 535 540 Asp Leu Leu Glu Asp Asn Val Thr Pro IleAsp Thr Thr Ile Met Ala 545 550 555 560 Lys Asn Glu Val Phe Cys Val GlnPro Glu Lys Gly Gly Arg Lys Pro 565 570 575 Ala Arg Leu Ile Val Phe ProAsp Leu Gly Val Arg Val Cys Glu Lys 580 585 590 Met Ala Leu Tyr Asp ValVal Thr Lys Leu Pro Leu Ala Val Met Gly 595 600 605 Ser Ser Tyr Gly PheGln Tyr Ser Pro Gly Gln Arg Val Glu Phe Leu 610 615 620 Val Gln Ala TrpLys Ser Lys Lys Thr Pro Met Gly Phe Ser Tyr Asp 625 630 635 640 Thr ArgCys Phe Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Thr Glu 645 650 655 GluAla Ile Tyr Gln Cys Cys Asp Leu Asp Pro Gln Ala Arg Val Ala 660 665 670Ile Lys Ser Leu Thr Glu Arg Leu Tyr Val Gly Gly Pro Leu Thr Asn 675 680685 Ser Arg Gly Glu Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val 690695 700 Leu Thr Thr Ser Cys Gly Asn Thr Leu Thr Cys Tyr Ile Lys Ala Arg705 710 715 720 Ala Ala Cys Arg Ala Ala Gly Leu Gln Asp Cys Thr Met LeuVal Cys 725 730 735 Gly Asp Asp Leu Val Val Ile Cys Glu Ser Ala Gly ValGln Glu Asp 740 745 750 Ala Ala Ser Leu Arg Ala Phe Thr Glu Ala Met ThrArg Tyr Ser Ala 755 760 765 Pro Pro Gly Asp Pro Pro Gln Pro Glu Tyr AspLeu Glu Leu Ile Thr 770 775 780 Ser Cys Ser Ser Asn Val Ser Val Ala HisAsp Gly Ala Gly Lys Arg 785 790 795 800 Val Tyr Tyr Leu Thr Arg Asp ProThr Thr Pro Leu Ala Arg Ala Ala 805 810 815 Trp Glu Thr Ala Arg His ThrPro Val Asn Ser Trp Leu Gly Asn Ile 820 825 830 Ile Met Phe Ala Pro ThrLeu Trp Ala Arg Met Ile Leu Met Thr His 835 840 845 Phe Phe Ser Val LeuIle Ala Arg Asp Gln Leu Glu Gln Ala Leu Asp 850 855 860 Cys Glu Ile TyrGly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro 865 870 875 880 Pro IleIle Gln Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser 885 890 895 TyrSer Pro Gly Glu Ile Asn Arg Val Ala Ala Cys Leu Arg Lys Leu 900 905 910Gly Val Pro Pro Leu Arg Ala Trp Arg His Arg Ala Arg Ser Val Arg 915 920925 Ala Arg Leu Leu Ala Arg Gly Gly Arg Ala Ala Ile Cys Gly Lys Tyr 930935 940 Leu Phe Asn Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Ala945 950 955 960 Ala Ala Gly Gln Leu Asp Leu Ser Gly Trp Phe Thr Ala GlyTyr Ser 965 970 975 Gly Gly Asp Ile Tyr His Ser Val Ser His Ala Arg ProArg Trp Ile 980 985 990 Trp Phe Cys Leu Leu Leu Leu Ala Ala Gly Val GlyIle Tyr Leu Leu 995 1000 1005 Pro Asn Arg 1010 20 amino acids amino acidsingle linear peptide 6 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg GlyArg Arg Gln Pro 1 5 10 15 Ile Pro Lys Ala 20 266 amino acids amino acidsingle linear peptide 7 Ala Val Asp Phe Ile Pro Val Glu Asn Leu Glu ThrThr Met Arg Ser 1 5 10 15 Pro Val Phe Thr Asp Asn Ser Ser Pro Pro ValVal Pro Gln Ser Phe 20 25 30 Gln Val Ala His Leu His Ala Pro Thr Gly SerGly Lys Ser Thr Lys 35 40 45 Val Pro Ala Ala Tyr Ala Ala Gln Gly Tyr LysVal Leu Val Leu Asn 50 55 60 Pro Ser Val Ala Ala Thr Leu Gly Phe Gly AlaTyr Met Ser Lys Ala 65 70 75 80 His Gly Ile Asp Pro Asn Ile Arg Thr GlyVal Arg Thr Ile Thr Thr 85 90 95 Gly Ser Pro Ile Thr Tyr Ser Thr Tyr GlyLys Phe Leu Ala Asp Gly 100 105 110 Gly Cys Ser Gly Gly Ala Tyr Asp IleIle Ile Cys Asp Glu Cys His 115 120 125 Ser Thr Asp Ala Thr Ser Ile LeuGly Ile Gly Thr Val Leu Asp Gln 130 135 140 Ala Glu Thr Ala Gly Ala ArgLeu Val Val Leu Ala Thr Ala Thr Pro 145 150 155 160 Pro Gly Ser Val ThrVal Pro His Pro Asn Ile Glu Glu Val Ala Leu 165 170 175 Ser Thr Thr GlyGlu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu 180 185 190 Val Ile LysGly Gly Arg His Leu Ile Phe Cys His Ser Lys Lys Lys 195 200 205 Cys AspGlu Leu Ala Ala Lys Leu Val Ala Leu Gly Ile Asn Ala Val 210 215 220 AlaTyr Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr Ser Gly Asp 225 230 235240 Val Val Val Val Ala Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp 245250 255 Phe Asp Ser Val Ile Asp Cys Asn Thr Cys 260 265 20 amino acidsamino acid single linear peptide 8 Asp Glu Met Glu Glu Cys Ser Gln HisLeu Pro Tyr Ile Glu Gln Gly 1 5 10 15 Met Met Leu Ala 20 20 amino acidsamino acid single linear peptide 9 Ile Glu Gln Gly Met Met Leu Ala GluGln Phe Lys Gln Lys Ala Leu 1 5 10 15 Gly Leu Leu Gln 20 20 amino acidsamino acid single linear peptide 10 Leu Ala Glu Gln Phe Lys Gln Lys AlaLeu Gly Leu Leu Gln Thr Ala 1 5 10 15 Ser Arg Gln Ala 20 20 amino acidsamino acid single linear peptide 11 Val Trp Ala Arg Pro Asp Tyr Asn ProPro Leu Val Glu Thr Trp Lys 1 5 10 15 Lys Pro Asp Tyr 20 20 amino acidsamino acid single linear peptide 12 Glu Thr Trp Lys Lys Pro Asp Tyr GluPro Pro Val Val His Gly Cys 1 5 10 15 Pro Leu Pro Pro 20 20 amino acidsamino acid single linear peptide 13 Val His Gly Cys Pro Leu Pro Pro ProLys Ser Pro Pro Val Pro Pro 1 5 10 15 Pro Arg Lys Lys 20

What is claimed is:
 1. A diagnostic reagent for detecting hepatitis Cvirus (HCV) infection comprising a solid phase to which three or moredifferent HCV antigen-carrier protein conjugates are bound, wherein: thecarrier protein and HCV antigen are present in each conjugate at a ratioof about 1:3 to 1:20 (carrier protein:hepatitis C virus antigen), eachHCV antigen-protein conjugate has only one type of HCV antigen bound toit, and each HCV antigen is from an antigen selected from the groupconsisting of core antigen, NS3 antigen, NS4 antigen and NS5 antigen. 2.A diagnostic reagent for hepatitis C virus (HCV) infection comprising asolid phase to which three or more different HCV antigen components arebound, wherein: at least one of the antigen components is from anantigen selected from the group consisting of core antigen, NS4 antigenand the NS5 antigen and is attached to a carrier protein to form a HCVantigen-protein conjugate, the bound carrier protein and HCV antigen arepresent in each component at a ratio of about 1:3 to 1:20 (carrierprotein:hepatitis C virus antigen), the HCV antigen-protein conjugatehas only one type of HCV antigen bound to it, and each HCV antigencomponent is from an HCV antigen selected from the group consisting ofcore antigen, NS3 antigen, NS4 antigen and NS5 antigen.
 3. Thediagnostic reagent for hepatitis C virus infection of claim 1 or 2,wherein each of the antigens of the three or more antigen-carrierprotein conjugates has one or more different epitopes having HCVantigenic activity.
 4. The diagnostic reagent for hepatitis C virusinfection of claim 1 or 2, wherein the carrier protein is selected fromthe group consisting of bovine serum albumin (BSA), ovalbumin andhemocyanin.
 5. The diagnostic reagent for hepatitis C virus infection ofclaim 1, wherein the carrier protein is selected from the groupconsisting of BSA, ovalbumin and hemocyanin.
 6. The diagnostic reagentfor hepatitis C virus infection of claim 1 or 2 wherein the solid phaseis carrier particles.
 7. The diagnostic reagent for hepatitis C virusinfection of claim 6, wherein the carrier particles are hydrophobicparticles.
 8. The diagnostic reagent for hepatitis C virus infection ofclaim 7, wherein the hydrophobic particles are polystyrene latex.
 9. Amethod of diagnosing the presence or absence of hepatitis C virusinfection, comprises a) contacting the diagnostic reagent for hepatitisC virus infection according to claim 1 or 2 with a sample, b) incubatingthe contacted sample under agglutinating conditions, and c) measuringthe degree of agglutination of the carrier particles, whereby the degreeof agglutination is correlated with the presence or absence of hepatitisC virus infection.
 10. The method of diagnosing the presence or absenceof hepatitis C virus infection of claim 9, wherein the degree ofagglutination is measured by a flow cytometer.
 11. The method ofdiagnosing the presence or absence of hepatitis C virus infection asclaimed in claim 10, wherein the measurement by flow cytometry is madeby measuring forward-scattered light.